The formulation of .alpha.-thrombin is a central event during the blood coagulation cascade. Prothrombin is activated to .alpha.-thrombin by the prothrombinase complex which is composed of the serine protease factor Xa and the protein cofactor factor Va assembled on a membrane surface in the presence of Ca.sup.2+ ions (1).sup.1. In the absence of factor Va, factor Xa is capable of activating prothrombin to .alpha.-thrombin (2); however, the prothrombinase complex is 5 orders of magnitude more efficient in generating .alpha.-thrombin than factor Xa acting along (2). Once formed, .alpha.-thrombin acts as a procoagulant enzyme, catalyzing the formulation of the fibrin clot by converting soluble fibrinogen to insoluble fibrin polymers (3). .alpha.-Thrombin also binds to the endothelial cell receptor thrombomodulin, and the resulting .alpha.-thrombin-thrombomodulin complex exhibits altered proteolytic specificity (4,5). This complex catalyzes the activation of protein C to form the anticoagulant enzyme activated protein C (APC)..sup.1 FNT .sup.1 Reference articles are indicated herein by (number) and are identified before the claims under the heading CITED REFERENCES.
Activation of bovine protein C occurs by cleavage of a single peptide bond (Arg.sup.171) located at Arg.sup.14 of the heavy chain of protein C (5). Bovine protein C is also activated by the factor Xz-thrombomodulin complex which produces an APC molecule (6). The proteolytic APC antithrombotic function is expressed by inactivating factor Va and factor VIIIa, two important cofactors of the blood coagulation cascade (7, 8).
Factor V circulates s a single chain procofactor (M.sub.4 330,000). The CDNA sequence for bovine factor V and deduced amino acid sequence have been determined (9). The active form of the protein, factor Va (10-12), is composed of a heavy chain (M.sub.r 94,000) containing the NH2-terminal part of the procofactor (residues 1-713) and a light chain (Mr 74,000) containing the COOH-terminal part of the molecule (amino acids 1537-2183; Ref. 9). The two chains are noncovalently associated (13, 14).
The inactivation of factor Va by APC occurs in the presence as well as in the absence of a lipid bilayer or platelet surface (15-20). Previous studies have demonstrated that bovine factor Va in the absence of a lipid bilayer and in the presence of high concentrations of APC is slowly and partially inactivated (20). In contrast, earlier kinetic data showed that in the presence of an anionic phospholipid membrane surface factor Va is rapidly and completely inactivated by APC (15-17). Recent data also showed that bovine factor Va phosphorylation at the COOH-terminal end of the heavy chain on Ser.sup.690 results in a molecule that is more sensitive to APC inactivation that its native counterpart (21). The identification of the APC proteolytic cleavage sites in the cofactor observed in the absence of phospholipid has been correlated with the partial inactivation of factor Va. Thus, although studies demonstrate that inactivation of the cofactor is a rapid phenomenon in the presence of a membrane surface (15-17), the chemical studies relating the APC effect on the factor Va molecule functions have been performed in the absence of lipids (18,22,23).
APC cleavage of the bovine factor Va heavy chain at Arg.sup.505 and Arg.sup.662 (and Arg.sup.506 in the human sequence) is responsible for the partial inactivation of the cofactor (20). However, in these studies even after prolonged incubation of factor Va with high APC concentrations (i.e., 4 h, 1:1 molar ratio) in the absence of a membrane surface the cofactor still retained 30-35% of its initial cofactor activity (20). Thus, the contribution of the membrane to the mechanism by which APC inactivates factor Va has not been elucidated yet.
It has been shown that vitamin K-dependent protein S plays a role in the APC inactivation process only in the presence of a phospholipid membrane (24,25). Its functions have not been established clearly yet, but protein S must play an important role as an antithrombotic agent since studies demonstrated that familial heterozygous deficiency of protein S is associated with venous and arterial thrombosis (26,27).